Mast cells and their mediators play critical roles in homeostatic wound healing pathways. Therefore, the premature demise of the mast cell population via programmed cell death or apoptosis may permit uncontrolled tissue remodeling which progresses to fibrosis. Metalloproteinases and their inhibitors exert diverse influences on cellular functions by processing matrix proteins and by regulating the dynamic display of membrane- anchored receptors, adhesion molecules and growth factors at the cell surface. The overall hypothesis of this proposal is that metalloproteinase-dependent mechanisms regulate mast cell apoptosis during fibrinogenesis. Binding of c-kit ligand (stem cell factor) to kit receptor tyrosine kinase regulates mast cell proliferation and apoptosis, and also induces receptor down-regulation via an uncharacterized metalloproteinase-dependent cleavage of Kit's extracellular domain. The experimental design employs complementary approaches using a rat model of lung transplant-associated airway fibrosis to study the role of metalloproteinases in mast cell apoptosis in vivo, and cultured mast cells to investigate the role of metalloproteinases and tissue inhibitors of metalloproteinases (TIMPs) in molecular mechanisms which regulate proteolytic shedding of the Kit ectodomain. Consistent features of mast cells in fibrotic lung disorders, including transplant-associated obliterative bronchiolitis are fluctuations in the size of their population and persistent degranulation of stored mediators. To address the overall hypothesis, the proposed studies will investigate metalloproteinase- dependent induction of mast cell apoptosis and the role of mast cell mediators in regulating proteolytic processing of Kt receptor. Aim 1 will identify the role of metalloproteinases and mast cell apoptosis during the development of obliterative bronchiolitis in a rat lung allograft model. Aim 2 will clarify the metalloproteinase-dependent Kit receptor shedding mechanism by investigating mast cell expression of metalloproteinases such as tumor necrosis. Factor-alpha converting enzyme (TACE), and by identifying the proteases and peptide sites cleaved in Kit ectodomain processing. Aim 3 will determine the role of mast cell proteases and oxygen radicals in proteolytic and non-proteolytic processing of TIMPs which not only inhibit metalloproteolytic sheddases, but also regulate apoptosis. These studies should provide valuable insights into the role of mast cells in the pathogenesis of fibrotic lung disorders and also clarify mechanisms responsible for the development of obliterative bronchiolitis, the major obstacle to improved outcome and survival in lung transplant patients.